Chiral separations of amino acids

ABSTRACT

An enantiomeric mixture of a chiral amino acid is separated into its respective enantiomers through chromatography on a chiral polysaccharide stationary phase eluting with a mobile phase comprising (i) a liquid lower alkanol and (ii) a carboxylic acid soluble in the lower alkanol. The mobile phase may also contain a liquid hydrocarbon.

This application claims benefit of provisional application No.60/040,987, filed Mar. 18, 1997.

The present invention relates to the separation of chiral materialsutilizing high performance liquid chromatography (HPLC) techniques.

It is know that the addition of an acidic modifier such astrifluoroacetic acid (TFA) to the mobile phase is required for theseparation of certain carboxylic acids. It also is know that moleculeswhich contain an amino group may require the addition of triethylamine(TEA) and/or diethylamine (DEA) as modifiers in order to obtainsatisfactory resolution and peak shape. DEA or TEA acts as competitorswith the basic analyte toward the basic silane groups on the silicasupport.

Tang, J Chirality, (1996) reports on the use of TFA as a mobile phasemodifier for the separation of basic compounds.

DETAILED DESCRIPTION

The present invention pertains to a method of separating an enantiomericmixture of a chiral compound containing an amino nitrogen atom, or asalt thereof, specifically an amino acid, into its respectiveenantiomers. This method comprises subjecting the chiral mixture tochromatography on chiral polysaccharide stationary phase. The stationaryphase is then eluted with a mobile phase comprising (i) a liquidhydrocarbon, (ii) a liquid lower alkanol, and (iii) a carboxylic acidsoluble in the liquid hydrocarbon and lower alkanol.

The enantiomeric HPLC separations encompassed by the present inventionutilize chiral polysaccharides as stationary phases. Typically these arearomatic carbamate or ester derivatives of cellulose or amylose whichcan be generically represented by the formula: ##STR1## in which thedepicted glucosidic linkage is either α (amylose) or β (cellulose).

The depicted R groups can be for example a phenylcarbamate orα-phenethylcarbamate, which itself is chiral, or a benzoate group.Typical R groups thus include 3,5-dimethylphenyl carbamate,α-phenethylcarbamate, and 4-methylbenzoate, e.g.: ##STR2##

Such chiral polysaccharide stationary supports are commerciallyavailable from Chiral Technologies, Inc., Exton, Pa., under thetrademarks CHIRALPAK® amylosic stationary phase and CHIRALCEL®cellulosic stationary phase. Suitable materials include CHIRALPAK® AD™,an amylose derivative in which each glucose monomer carries three3,5-dimethylphenyl carbamate groups, CHIRALPAK® AS™, an amylosederivative in which each glucose monomer carries three(S)-α-phenethylcarbamate groups, CHIRALCEL® OD™, a cellulose derivativein which each glucose monomer carries three 3,5-dimethylphenyl carbamategroups, and CHIRALCEL® OJ™, a cellulose derivative in which each glucosemonomer carries three 4-methylbenzoyl groups. Reference may be made toU.S. Pat. Nos. 4,912,205 and 5,434,299 for further details, thedisclosures of which are incorporated herein by reference. Amylosederivatives, particularly CHIRALPAK® AD™, are preferred.

The stationary phase conveniently can be packed in columns adapted foruse with commercially available HPLC systems, as for example thoseavailable from Shimadzu, Columbia, Md., and Jasco, Easton, Md. Generallythe particle diameter will be from about 1 to about 100 μm, typicallyfrom about 5 to about 75 μm. Multiple or single columns can be employed.A simulated moving bed apparatus also can be employed, as described forexample in U.S. Pat. Nos. 5,434,298, 5,434,299, 5,456,825, and5,498,752, the disclosures of which are incorporated herein byreference.

The eluent or mobile phase comprises (i) a liquid lower alkanol, and(ii) a strong carboxylic acid soluble in the lower alkanol. The loweralkanol can contain from 1 to 6 carbon atoms such as methanol, ethanol,n-propanol, isopropanol, butanol, and the like, but preferable isethanol or methanol. In some instances separation is poor withisopropanol but is satisfactory when the mobile phase employs ethanol ormethanol.

In addition to the lower alkanol and a strong carboxylic acid, theeluent or mobile phase can contain a liquid hydrocarbon, as for examplepentane, hexane, or heptane.

Gradient mixtures often will be employed; e.g., starting with apredominantly hydrocarbon mixture {such as 70:30 hydrocarbon:alkanol(v/v)} and progressively increasing the amount of alkanol to as high as100% alkanol. The carboxylic acid can be any relatively strong acidwhich is soluble in the lower alkanol (and any liquid hydrocarbon whichis present), as for example formic acid, acetic acid, a substitutedacetic acid, propionic acid, and the like. Particularly preferred aretrihaloacetic acids such as trifluoroacetic acid and trichloroaceticacid.

Without wishing to be bound by any theory of operation, it appears oneof the enantiomers is retained at about the same retention whether ornot the carboxylic acid is present, while the other enantiomer shows asignificant increase in retention times when the carboxylic acid ispresent, thereby facilitating separation. It further appears the that atlow pH, the amine group is protonated by the carboxylic acid with theanalytes separating as ion pairs.

In one embodiment, the mobile phase also will contain a secondary ortertiary amine. This is present in less than a 1:1 molar ratio to thecarboxylic acid. Preferred amines are di-(lower alkyl) amine andtri(lower alkyl)amine such as diethylamine and triethylamine. Additionof these amines to the mobile phases produces several additionalbenefits. The addition of certain carboxylic acids to the mobile phasecan increase UV absorbance, leading to detection problems for analytesof low UV absorbance. Addition of an amine to the mobile phase, however,decreases the absorbance, thus facilitating the monitoring of theanalyte elution by UV absorbance at less than 240 nm. Second, theaddition of the amine can lead to significant decrease in peak tailingfor basic analytes. The carboxylic acid still acts as an ion pair agent,but the amine salt complex competes with active sites on the chiralcolumn which can cause peak tailing. This decrease in peak tailing cansignificantly improve the resolution.

The present method is suitable for separating chiral amino acidsincluding both naturally occurring α-amino acids, such as alanine,arginine, lysine, phenylalanine, serine, valine, threonine, glutamicacid, DOPA, norleucine, leucine, norvaline and the like, as well as β-,γ-, and ω-amino acids. Amino acids normally exist as Zwitter ions inneutral solution so that both its carboxylic acid and amino group areionized. Consequently, although these molecules are very insoluble intypical mobile phases, they can be separated on the polysaccharidephases through appropriate pH adjustment and control of the ion-pairingagent.

The process also can be used with a derivative of an amino acid such asa carbamate such as an N-tert.-butyloxycarbonyl or benzyloxycarbonylderivative. Oxycarbonyl derivatives are useful since they tend to bemore soluble than the free amine in the mobile phase. Typical of suchderivatives are N-tert.-butyloxycarbonylproline,N-tert.-butyloxycarbonyltyrosine, andN-tert.-butyloxy-carbonyltryptophane. An acid modifier is used in thiscase to keep the derivatized amino acid neutral.

The process also can be used with chiral amines which are not aminoacids including aliphatic, cycloaliphatic, aromatic, and heterocyclicamines. The amine can be a primary, secondary, tertiary, or quaternaryamine. Typical amines for which the present process is suitable includewithout limitation albuterol, metoprolol, propranolol, pindolol, andother propanol amines, methadone, 1-methyl-1-phenylethane,2-aminobutane, 2-amino-1-butanol, 1-amino-1-phenylethane,1-amino-1-(2-methoxy-5-fluorophenyl)ethane, 1-amino-1-phenyl-propane,1-amino-1-(4-hydroxyphenyl)propane, 1-amino-1-(4-bromophenyl)-propane,1-amino-1-(4-nitrophenyl)propane, 1-phenyl-2-aminopropane,1-(3-tri-fluoromethylphenyl)-2-aminopropane, 2-aminopropanol,1-amino-1-phenyl-butane, 1-phenyl-2-aminobutane,1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane,1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-2-methylcyclopentane,1-amino-3-methylcyclopentane, 1-amino-2-methylcyclohexane, aminophenols,and 1-amino-1-(2-naphthyl)ethane.

The separation will be conducted at ambient temperatures; e.g., 25-40°C. pH will vary depending upon the nature of the material beingchromatographed but generally will be from about 2 to about 7. Typicalflow rates are from about 0.2 mL/min. to about 25 mL/min., depending onthe apparatus, column dimensions, and stationary phase.

Separation can be monitored by measuring the optical activity of theeluted material, using for example a device such as the IBZ Chiralyser®instrument (available from JM Science, Inc., Grand Island, N.Y.) whichmonitors the rotation of plane polarized light. The refractive index,evaporative light scattering, and UV detectors may be used asalternative monitors. The parameter selected for detection will dependon the specific material being eluted.

The following examples will serve to further typify the nature of theinvention but should not be construed as limitation on the scope thereofwhich is defined solely by the appended claims.

EXAMPLE 1

A racemic mixture of norleucine was separated into its two enantiomersusing CHIRALPAK® AD™, an amylose derivative in which each glucosemonomer carries three 3,5-dimethylphenyl carbamate groups, as thestationary phase and 93:7:0.2 heptane:ethanol:trifluoroacetic acid asthe mobile phase. The flow rate was 1 mL/min. and detection was measuredat 225 nm.

EXAMPLE 2

A racemic mixture of leucine was separated into its two enantiomersusing CHIRALPAK® AD™ as the stationary phase and 93:7:0.2heptane:ethanol:trifluoroacetic acid as the mobile phase. The flow ratewas 1 mL/min. and detection was measured at 225 nm.

EXAMPLE 3

A racemic mixture of norvaline was separated into its two enantiomersusing CHIRALPAK® AD™ as the stationary phase and 93:7:0.2heptane:ethanol:trifluoroacetic acid as the mobile phase. The flow ratewas 1 mL/min. and detection was measured at 225 nm.

EXAMPLE 4

A racemic mixture of N-tert.-butyloxycarbonyltyrosine was separated intoits two enantiomers using CHIRALPAK® AD™ as the stationary phase and85:15:0.2 heptane:ethanol:trifluoroacetic acid as the mobile phase. Theflow rate was 1 mL/min. and detection was measured at 240 nm.

EXAMPLE 5

A racemic mixture of N-tert.-butyloxycarbonyltryptophane was separatedinto its two enantiomers using CHIRALPAK® AD™ as the stationary phaseand 85:15:0.2 heptane:ethanol:trifluoroacetic acid as the mobile phase.The flow rate was 1 mL/min. and detection was measured at 240 nm.

EXAMPLE 6

A racemic mixture of N-tert.-butyloxycarbonylproline was separated intoits two enantiomers using CHIRALPAK® AD™ as the stationary phase and93:7:0.2 heptane:ethanol:trifluoroacetic acid as the mobile phase. Theflow rate was 1 mL/min. and detection was measured at 235 nm.

EXAMPLE 7

As note the process can be practiced with chiral amines which are notamino acids, as following illustrates. A chiral mixture of1-(3-hydroxymethyl-4-hydroxyphenyl)-2-(tert.-butylamino)-ethan-1-ol waschromatographed on a stationary phase of CHIRALCEL® OJ™, a cellulosederivative in which each glucose monomer carries three 4-methylbenzoylgroups, at room temperature at a flow rate of 1 mL/min., utilizing93:7:0.1 hexane:ethanol:diethylamine as the mobile phase. Separation wasmeasured by absorption at 230 nm. Only a single peak was observed. When95.5:4.5:0.1:0.17 hexane:ethanol:trifluoroacetic acid:triethylamine wasutilizing as the mobile phase, separation into two distinct peaks wasobserved.

EXAMPLE 8

1-(Naphth-1-yloxy)-3-(isopropylamino)-propan-2-ol was separated into itstwo enantiomers using CHIRALCEL® OD™, a cellulose derivative in whicheach glucose monomer carries three 3,5-dimethylphenyl carbamate groups,as the stationary phase and 80:20:0.2:0.34hexane:ethanol:trifluoroacetic acid:triethylamine as the mobile phase.

EXAMPLE 9

Following the procedure of example 8, 1-[4-(2-methoxyethyl)phenoxy]-3-(isopropylamino)-propan-2-ol was separated into its twoenantiomers using CHIRALCEL® OD™ as the stationary phase and80:20:0.2:0.34 hexane:ethanol:trifluoroacetic acid:triethylamine as themobile phase

What is claimed is:
 1. The method of separating an enantiomeric mixtureof a chiral amino acid, or a salt or derivative thereof, into therespective enantiomers which comprises subjecting said mixture tochromatography on chiral polysaccharide stationary phase eluting with amobile phase comprising (i) a liquid lower alkanol and (ii) a carboxylicacid soluble in the liquid hydrocarbon and lower alkanol.
 2. The methodof claim 1 wherein said carboxylic acid is acetic acid or a substitutedacetic acid.
 3. The method of claim 2 wherein said carboxylic acid is atrihaloacetic acid.
 4. The method of claim 3 wherein said trihaloaceticacid is trifluoroacetic acid.
 5. The method of claim 1 wherein saidlower alkanol is ethanol.
 6. The method of claim 1 wherein said loweralkanol is methanol.
 7. The method of claim 1 wherein said mobile phasecomprises a tertiary amine.
 8. The method of claim 7 wherein saidtertiary amine is tri-(lower alkyl) amine.
 9. The method of claim 8wherein said tertiary amine is triethylamine.
 10. The method of claim 1wherein said chiral polysaccharide stationary phase is an amylosederivative.
 11. The method of claim 10 wherein in which each glucosemonomer of said amylose derivative carries three 3,5-dimethylphenylcarbamate groups.
 12. The method of claim 1 wherein said amino acidderivative is a carbamate derivative.
 13. The method of claim 12 whereinsaid carbamate derivative is a N-tert.-butyloxycarbonyl derivative. 14.The method of claim 1 wherein said mobile phase also contains a liquidhydrocarbon.
 15. The method of claim 14 wherein said hydrocarbon ispentane, hexane, or heptane.
 16. The method of separating a mixture of achiral amino acid, or a salt or derivative thereof, into the respectiveenantiomers which comprises subjecting said mixture to chromatography onchiral polysaccharide stationary phase with a mobile phase comprising(i) a liquid hydrocarbon, (ii) ethanol, and (iii) a trifluoroaceticacid.